Premature prostaglandin F2alpha secretion causes luteal regression in GnRH-induced short estrous cycles in cyclic dairy heifers

This study aimed to confirm that the luteolysis in normal-cycling dairy heifers seen during short estrous cycles induced with cloprostenol (Clp) and GnRH administered 24h apart is caused by a premature release of prostaglandin F(2alpha) (PGF(2alpha)). A further aim was to study the PGF(2alpha) release pattern more closely to determine whether it resembles the spontaneous release occurring during normal regression of the corpus luteum (CL) or whether PGF(2alpha) is continuously secreted after the induced ovulations, leading to short estrous cycles. Twenty-four Ayrshire heifers were allotted to four equally sized groups. After estrus synchronization with 0.5mg of Clp, a new luteolysis was induced with 0.5mg of Clp on Day 6 (groups T-d6 and C-d6) or Day 7 (groups T-d7 and C-d7) after ovulation. Gonadorelin (0.1mg i.m.) was given to groups T-d6 and T-d7 to induce premature ovulation 24h later. Groups C-d6 and C-d7 served as controls. Ovaries were examined daily by transrectal ultrasonography, while blood samples (for progesterone and 15-ketodihydro-PGF(2alpha) analyses) were obtained via a jugular catheter every 3h, starting from the second Clp treatment and continuing for 9 days postovulation. Unresponsiveness to Clp or anovulation resulted in 4 C-d6 heifers being excluded. Four heifers in group T-d6 and three in group T-d7 had a short estrous cycle of 8-12 days, while all others had a cycle of normal length. Significant elevations in 15-ketodihydro-PGF(2alpha) concentrations with recurrent high peaks coincided with a decrease in progesterone concentration and were detected in all heifers that showed a short estrous cycle, but not in any heifers with normal estrous cycles in groups T and C. In conclusion, a premature release of PGF(2alpha), which closely resembles its release during spontaneous luteolysis, causes luteal regression in these short cycles.     

Rapid isolation of yeast genomic DNA: Bust n' Grab

Background

Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.

Results

Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC.

Conclusion

An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

     

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

Background

Claudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.

Methods

47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.

Results

Claudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.

Conclusion

Claudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.     

Selected ion monitoring in quantitative gas-liquid chromatographic-mass spectrometric detection of fatty acid methyl esters from environmental samples

To calculate selected ion monitoring (SIM) gas-liquid chromatography (GLC)-mass spectrometry (MS) results of phospholipid fatty acids (PLFAs) from environmental samples, coefficients were calculated for each fatty acid by dividing the sum of ion intensities in SCAN with that of ions followed in SIM. The SIM chromatogram areas were multiplied with the coefficients, and then processed as in SCAN. The results were compared to those obtained using calibration curves and SCAN. The calibration curve and coefficient based results had the greatest errors of 7.8 and 6.7%, respectively, outside standard deviations of SCAN percentages. The PLFA contents calculated using calibration curves and coefficients were 104.9+/-7.3% and 101.5+/-8.6%, respectively, of SCAN values. SIM increased sensitivity approximately 10-fold from SCAN, and the smallest detectable injected amount was approximately 50 ng (0.18 nmol) for 20 fatty acids, corresponding to 4 x 10(6) cells.     

Mucosa-Associated Bacteria in the Human Gastrointestinal Tract Are Uniformly Distributed along the Colon and Differ from the Community Recovered from Feces

The human gastrointestinal (GI) tract harbors a complex community of bacterial cells in the mucosa, lumen, and feces. Since most attention has been focused on bacteria present in feces, knowledge about the mucosa-associated bacterial communities in different parts of the colon is limited. In this study, the bacterial communities in feces and biopsy samples from the ascending, transverse, and descending colons of 10 individuals were analyzed by using a 16S rRNA approach. Flow cytometric analysis indicated that 10⁵ to 10⁶ bacteria were present in the biopsy samples. To visualize the diversity of the predominant and the Lactobacillus group community, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was performed. DGGE analysis and similarity index comparisons demonstrated that the predominant mucosa-associated bacterial community was host specific and uniformly distributed along the colon but significantly different from the fecal community (P < 0.01). The Lactobacillus group-specific profiles were less complex than the profiles reflecting the predominant community. For 6 of the 10 individuals the community of Lactobacillus-like bacteria in the biopsy samples was similar to that in the feces. Amplicons having 99% sequence similarity to the 16S ribosomal DNA of Lactobacillus gasseri were detected in the biopsy samples of nine individuals. No significant differences were observed between healthy and diseased individuals. The observed host-specific DGGE profiles of the mucosa-associated bacterial community in the colon support the hypothesis that host-related factors are involved in the determination of the GI tract microbial community.     

Using incidental mark‐encounter data to improve survival estimation

1. Obtaining robust survival estimates is critical, but sample size limitations often result in imprecise estimates or the failure to obtain estimates for population subgroups. Concurrently, data are often recorded on incidental reencounters of marked individuals, but these incidental data are often unused in survival analyses.
2. We evaluated the utility of supplementing a traditional survival dataset with incidental data on marked individuals that were collected ad hoc. We used a continuous time‐to‐event exponential survival model to leverage the matching information contained in both datasets and assessed differences in survival among adult and juvenile and resident and translocated Mojave desert tortoises (Gopherus agassizii).
3. Incorporation of the incidental mark‐encounter data improved precision of all annual survival point estimates, with a 3.4%–37.5% reduction in the spread of the 95% Bayesian credible intervals. We were able to estimate annual survival for three subgroup combinations that were previously inestimable. Point estimates between the radiotelemetry and combined datasets were within |0.029| percentage points of each other, suggesting minimal to no bias induced by the incidental data.
4. Annual survival rates were high (>0.89) for resident adult and juvenile tortoises in both study sites and for translocated adults in the southern site. Annual survival rates for translocated juveniles at both sites and translocated adults in the northern site were between 0.73 and 0.76. At both sites, translocated adults and juveniles had significantly lower survival than resident adults. High mortality in the northern site was driven primarily by a single pulse in mortalities.
5. Using exponential survival models to leverage matching information across traditional survival studies and incidental data on marked individuals may serve as a useful tool to improve the precision and estimability of survival rates. This can improve the efficacy of understanding basic population ecology and population monitoring for imperiled species.